Project Summary The directed beating of motile cilia is a critical aspect of tissue function in a variety of developmental and physiological contexts including proper neural development, egg migration through the oviduct and mucus clearance in the respiratory tract. The loss of cilia motility results in a wide range of phenotypes including hydrocephaly, infertility, situs inversus, and respiratory dysfunction. We have developed the ciliated epithelium of Xenopus larval skin as a model system to ask: How do ciliated cells generate hundreds of cilia and how do they orient those cilia in an organized way? We have developed confocal light microscopic methods for visualizing specific aspects of ciliated cells in the developing skin of Xenopus embryos. These methods allow us to visualize the massive centriole duplication required to generate the approximately 150 basal bodies that nucleate the cilia. Additionally, we can visualize and accurately quantify the cytoskeletal interactions that facilitate the establishment of cilia orientation. Using these methods we will address: (1.) The regulation of microtubule dynamics during the polarization of ciliated epithelia, (2.) The regulation of actin dynamics during the polarization of ciliated epithelia, and (3.) The regulation of centriole amplification. Our results will provide a long sought after missing link between polarity cues and the regulation of cytoskeletal dynamics during cellular polarization. While our work is focused on ciliated epithelia, it will provide a clear understanding of the downstream regulation of polarity cues that is important in numerous developmental and disease contexts. Additionally, defects in centriole duplication highly correlate with late stage cancer progression, indicating an uncoupling of duplication from the cell cycle. Our work will address the question of how centrioles can be generated in the absence of the cell cycle cues.